The present invention relates to catalysts for the cleavage of ester bonds, in particular, phosphate ester bonds related to a biological system. An effective catalyst would have the following properties:
1) Efficiency at physiological temperature and pH; PA1 2) Specificity for certain biological substrates; PA1 3) Low toxicity for physiological systems; and PA1 4) Easy administration to a biological system and ready subsequent elimination.
Various biologically important phosphate esters may be hydrolyzed according to the methods of the present invention, including RNA, DNA, phospholipids, phosphate anhydrides, UDP-glucose or a widely used substrate for assays, p-nitrophenylphosphate ester.
Many divalent and trivalent metal salts have been shown to promote the hydrolysis of phosphate ester bonds. Komiyama et al. (1992) reported the hydrolysis of adenylyl(3'-5')adenosine and uridyl(3'-5')uridine at pH 8.0, 30.degree. C. by rare earth metal(III) ions. A Cerium(III) hydroxide cluster has been reported to hydrolyze 3',5'-cyclic adenosine monophosphate (Sumaoka, et al. 1992). Browne and Bruice (1992) reported the hydrolysis of bis(8-hydroxyquinoline)phosphate in the presence of divalent cations. However, in order to convey a degree of specificity to catalysis by the metal ion, complexes of metals with various ligands have been studied. The ligand may serve a number of roles in catalysis including, modulation of catalytic efficiency, and maintenance of the metal ion in solution, while also allowing for coupling of reagents having a binding specificity for a desired substrate.
Ligands complexing metal ions for use in the hydrolysis of phosphate ester bonds include: tris(aminopropyl)amine (trpn), 1,4,7,10-tetraazacyclododecane (cyclen), tris(2-aminoethyl)amine (tren), triethylenetetramine (trien), tetraethylenepentamine (tetren), bipyridine conjugates, imidazole, cyclodextrin derivatives, lysine, terpyridine (trpy), 1,2-diaminoethane, a bis(diaquo) complex, "metallomicelles" and a phenanthrolinepolyamine tridentate complex (Basile et al. 1987, Menger et al. 1987, Chung et al. 1990, Hendry and Sargeson, 1989, Shelton and Morrow, 1991, Ranganathan et al. 1993, Breslow and Huang, 1991, Modak et al. 1991, Kim and Chin, 1992, Chin et al. 1989, Chin and Banaszczyk, 1989a,b, Chin and Zou, 1987).
In order for a metal complex to function catalytically in vivo, the complex should not release bound metal ion. Morrow et al. (1992) have studied the cleavage of RNA by a lanthanide(III) hexamine Schiff-base (HAM) macrocyclic complex. Cleavage of the dinucleotide adenylyl-3',5'uridine 3'-monophosphate (ApUp) or of oligomers of adenylic acid (A12-A18) was reported at 37.degree. C. after 4 hours by several lanthanide complexes. Other hexadentate ligands such as EDTA formed lanthanide(III) complexes that are completely inactive in RNA cleavage under similar conditions. Inertness of the macrocyclic complex to metal release was reported to change dramatically throughout the lanthanide series. These complexes have some serious disadvantages, including high toxicity of the HAM ligand, weak coordination and dissociation of the lanthanide metals. Further, the ligand cannot be easily modified which precludes the generation of derivatives with substrate specificity.
Given the limitations of the HAM complex, it is clear that the development of new macrocycles, capable of chelating lanthanide metals and forming stable complexes which are able to cleave RNA, would be of utility.